The invention is related to reducing non-specific amplification in polymerase chain reactions. Specifically, the invention relates to the use of sorbitol and dimethylsulfoxide (DMSO) in polymerase chain reactions in an amount effective to increase the yield of target molecules.
The polymerase chain reaction (PCR) has greatly advanced the field of molecular biology by allowing the amplification and analysis of specific fragments of DNA. While simple in principle, PCR is prone to several types of artifacts that can frustrate analysis. For example, observed non-specific amplification of fragments may result from one or both of the primers binding to a sequence other than the target sequence, and produce one or more fragments of DNA that are not the desired product.
Non-specific amplification of DNA is often a problem in the amplification of conserved sequences, such as ribosomal DNA. Ribosomal RNA (rRNA) is by far the most abundant species of RNA present in a cell, typically representing 85-90% of the total RNA in a cell. rRNA is encoded by ribosomal DNA (rDNA). Each subunit of rRNA is encoded by a separate rDNA, although multiple rRNA genes exist in most organisms. The mitochondrion of eukaryotes and the chloroplast of plants also contain their own rRNA genes.
Ribosomal RNA has been used in hybridization studies for genetic analysis, evolution studies and taxonomic classification. However, rRNA sequences are at least partially similar in widely different organisms, and nearly all of the rRNA gene sequences from closely related organisms cross-hybridize. In PCR studies, specific amplification of rDNA sequences is difficult due to the relatedness of the sequences. Often, amplification of rDNA in PCR results in non-specific amplification, greatly complicating analysis.
In accordance with some embodiments of the methods of the invention, methods of reducing non-specific amplification of DNA in a polymerase chain reaction are provided comprising the steps of:
(a) providing a sample comprising a target DNA sequence of interest;
(b) contacting said sample with at least one enzyme having nucleic acid polymerase activity; and
(c) incubating said sample with said enzyme for a time and under conditions sufficient to amplify said target DNA sequence, forming amplified target DNA sequence;
wherein said incubation is performed in the presence of an amount of sorbitol, or sorbitol and DMSO effective to reduce said non-specific amplification relative to the amount of non-specific amplification observed in the absence of sorbitol, or sorbitol and DMSO.
Also provided in some embodiments are methods of amplifying ribosomal DNA in a polymerase chain reaction comprising the steps of:
(a) providing a sample comprising a ribosomal DNA target sequence of interest; and
(b) amplifying at least one nucleobase sequence of said ribosomal DNA to form amplified ribosomal DNA in a mixture of total amplified product;
wherein said amplification is performed in the presence of a sufficient amount of sorbitol and DMSO to reduce non-specific amplification relative to the amount of non-specific amplification observed in the absence of said sorbitol and said DMSO.
In some embodiments of the methods of the invention, methods are provided for detecting bacteria in a sample comprising: providing a sample comprising nucleic acid, said nucleic acid comprising at least one ribosomal DNA sequence; and amplifying at least one nucleobase sequence of said nucleic acid, thereby forming an amplified product, wherein said amplification is performed in the presence of an amount of sorbitol effective in reducing non-specific amplification relative to the amount of non-specific amplification observed in the absence of sorbitol. The amplification step may also include an effective amount of DMSO in combination with the sorbitol.
In some embodiments of the methods of the invention, sorbitol may be present in an amount of 0.05 M to 3.0 M. Alternatively, sorbitol may be present in an amount of 0.05 M to 2 M. In other embodiments, sorbitol may be present in an amount of 0.05 to 1 M. In other embodiments, sorbitol is added in an amount of 0.05 M to 0.75 M. In other embodiments, sorbitol may be present in an amount of 0.1 to 0.45 M. In other embodiments, sorbitol may be present in an amount of 0.2 M to 0.4 M. In other embodiments, sorbitol may be present in an amount of 0.25 M to 0.35 M.
In some embodiments of the methods of the invention, DMSO is present in an amount of 0.5% to 8.0%. In other embodiments, DMSO is present in an amount of 1.0% to 6.0%. In other embodiments, DMSO is present in an amount of 2.0% to 5.0%. In other embodiments, DMSO is present in an amount of 3.0% to 4.0%.
In some embodiments of the methods of the invention, DMSO is present in an amount of 1.25% and sorbitol is present in an amount of 0.15 M.
In some embodiments of the methods of the invention, non-specific amplification is reduced to less than 99%, 90%, 80%, 70%, 60%, 50% or 40%, 30%, or more of the amount of non-specific amplification obtained in the absence of sorbitol or sorbitol and DMSO.
In some embodiments of the methods of the invention, the amplified target sequence represents at least 50-70% of said total amplified product. In other embodiments, the amplified target sequence represents at least 70-90% of said total amplified product. In other embodiments, the amplified target sequence represents at least 90% of said total amplified product.
In certain embodiments, the methods of the invention are suitable for reducing non-specific amplification of DNA encoding ribosomal RNA.
The some embodiments of the methods of the invention, amplified products may be subsequently separated using a sieving or non-sieving medium. The nucleic acid sequence of the amplified products may be determined without or without prior separation.
The samples containing ribosomal DNA may be clinical samples such as blood, urine, cerebrospinal fluid, serum, saliva, mucus, skin, gastric secretions and/or stool.
In some embodiments of the methods of the invention, the amplification comprises contacting said nucleobase sequence with an enzyme having a polymerase activity. For example, the enzyme having polymerase activity may be selected from the group consisting of DNA polymerase from Thermus aquaticus, Thermus thermophilus, other Thermus species, Bacillus species, Thermococcus species, Thermotoga species, and Pyrococcus species. For example, suitable polymerases include AmpliTaq Gold(copyright) DNA polymerase; AmpliTaq(copyright) DNA Polymerase; AmpliTaq(copyright) DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Bst DNA polymerase large fragment from Bacillus stearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tma from Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu from Pyrococcus; and mutants, variants and derivatives thereof.
In some embodiments, the invention also provides compositions comprising:
(a) a nucleic acid sequence comprising a ribosomal DNA;
(b) at least two primers having a sequence that is complementary to a portion of said nucleic acid sequence adjacent to said ribosomal DNA;
(c) at least one enzyme having nucleic acid polymerase activity; and
(d) sorbitol or sorbitol and DMSO.
In other embodiments, the invention provides kits for the amplification of ribosomal DNA comprising, in one or more containers: an agent having polymerase activity, a plurality of deoxynucleotide triphosphates; and sorbitol, and, optionally, DMSO. The polymerase of the kit may be a DNA polymerase from Thermus aquaticus, Thermus thermophilus, other Thermus species, Bacillus species, Thermococcus species, Thermotoga species, and Pyrococcus species. For example, suitable polymerases include AmpliTaq Gold(copyright) DNA polymerase; AmpliTaq(copyright) DNA Polymerase; AmpliTaq(copyright) DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Bst DNA polymerase large fragment from Bacillus stearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tma from Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu from Pyrococcus; and mutants, variants and derivatives thereof.